Hepatitis B virus (HBV) infections represent a worldwide health problem with an estimated 300 million chronic carriers many of whom will develop cirrhosis and liver cancer. HBV is the seventh leading cause of death worldwide and the fourth leading cause of death due to infectious disease. Although an efficacious vaccine has been developed, it will be decades before its use will have an impact on the number of people dying from HBV- associated chronic liver disease and liver cancer. A precise understanding of the replication cycle may yield strategies to eliminate the carrier state. Although the replication cycle of HBV has been partially elucidated, many aspects remain unresolved. This is especially true for the protein-protein and protein-RNA interactions involved in genomic replication. This proposal will utilize our recently developed functional form of the HBV reverse transcriptase (pol) to elucidate aspects of pol function and structure with regard to the role of pol in genomic replication. The first specific aim is to define by mutagenesis the pol protein boundaries and conserved sequences involved in nucleotide priming, reverse transcription and RNA binding. The second specific aim is to define the RNA sequences involved in epsilon recognition, in nucleotide priming, and in strand transfer to DR1, as well as to determine if other pregenomic RNA domains are recognized by pol. Synthetic RNA templates will be used for in vitro pol assays to examine the preference of pol for cis versus trans templates for strand transfer reactions. The third aim is to define the role of pol phosphorylation in nucleotide priming, reverse transcription, RNA binding and the HBV replication cycle. The phosphorylation sites on pol will be mapped and mutated, and mutant pol proteins will be tested for in vitro pol activity and for complementation of HBV genomic replication. The fourth aim is to define the pol domains involved in core/pol interactions and the ability of core to influence in vitro pol activity. The fifth aim is to examine the role of pol associated proteins in pol function, and to identify the proteins that influence in vitro pol activity. The final aim is to collaborate in solving the crystallographic structure of pol and pol domains. Accomplishment of these goals will significantly increase our understanding of the central enzyme involved in HBV replication and our potential to eliminate HBV chronic infections.